Project Summary/Abstract Elucidating the early events on the B cell surface following antigen engagement of the B cell antigen receptor (BCR) can provide an assessment of the in vivo potential of an antigen (Ag) to drive B cell activation. In contrast to the ?clustering model?, the ?Dissociation Activation Model? (DAM) proposes an alternate conformational state of the resting BCR and how early events on the B cell membrane initiates B cell activation. In the DAM, B cell activation is dependent on the ability of an Ag to dissociate the inactive, resting BCR oligomers (BCR conformational opening), and promote nanoscale reorganization of BCR with co-stimulatory molecules, leading to full activation. Recent advances in HIV-1 Envelope (Env) protein designs have provided several key potential immunogens, however, how such viral Ags are sensed by BCRs on B cells that participates in anti-HIV-1 immune responses that generate broadly neutralizing antibodies (bnAbs) is not well understood. The overall objective of this proposal is to bridge the quantitative biophysical and membrane dynamics measurements of Ag-BCR interactions to ex-vivo and in-vivo B cell activation in the context of the DAM. Towards this goal, we will for the first time, study and define at nanoscale resolution, Ag-induced early BCR activation events and the costimulatory signaling required for full activation of B cells expressing BCRs specifying prototype HIV bnAbs or their precursors. We hypothesize that for optimal priming HIV Env immunogens, i.e. those capable of best activating B-cells and initiating robust affinity maturation, the exposure of the BCR to the Env antigens results first in nanoscale BCR conformational opening, followed by reorganization of BCRs and co-stimulatory molecules/kinases for signal amplification on the B cell membrane. In Aim 1, we will perform biophysical analyses and study B cell membrane dynamics to define the properties of HIV Env Ag-BCR interactions for optimal activation of B cells expressing bnAb or germline precursor BCRs. In Aim 2, we will use high resolution assays to determine Ag-binding properties that induce nanoscale BCR conformational opening, the role of costimulatory and regulatory molecules and the spatio-temporal reorganization of the B cell membrane following activation with HIV Env Ags. In Aim 3, we will test evaluated Env Ags for their ability to activate and drive affinity maturation in ?more amenable to induce?, (non-anergic or more ?mildly? anergic) bnAb B cells in nave knock-in (KI) repertoires. Results of such studies would be the first to allow a systematic method for predicting the potential of HIV immunogens to prime distinct bnAb precursors based on these defined Ag-induced early B cell activation events. In the long-term our studies will facilitate design and pre-selection of immunogens for testing in animal models and accelerate HIV-1 vaccine development.